Lipopolysaccharide (LPS) is a major cell wall component of gram-negative bacteria that potently stimulates inflammation. LPS associates with the circulating LPS binding protein (LBP) and through the CD14-Toll-Like Receptor (TLR) complex in immune cells like monocytes and macrophages, signals the production of many pro-inflammatory cytokines such as IL-Iβ, tumor necrosis factor-α (TNF-α) and IL-6. LPS also stimulates the production of chemokines such as IL-8 and monocyte chemotactic protein-1 (MCP-1). These cytokines and chemokines are important to host immunity and their over-expression can have deleterious consequences such as fatal septic shock. Although other organisms such as staphylococci and Candida can also cause this condition, the majority of the septic shock cases are due to infection by gram-negative bacilli (Bone, R. C. Gram-negative sepsis: a dilemma of modern medicine. Clinical Microbiology Reviews 1993, January; 6(1): 57-68; Paterson, R. L.; Webster, N. R. Journal of the Royal College of Surgeons of Edinburgh 2000, 45, 178-182). LPS stimulation through TLR4 results in activation of multiple signaling pathways in the cell, such as the JNK, p38, stress-activated mitogen-activated protein kinases as well as NFκB (Goodell et al., Journal of bacteriology 1985, 162, 391-397; Wright et al., Science 1990, 249, 1431-1433; Paik Y., Hepatology 2003, 37, 1043-1055). Synthetic glucocorticoids like dexamethasone (dex) are potent immunosuppressants and are used for prevention of LPS induced shock in animals (Mills et al., Proceedings of the Society for Experimental Biology and Medicine 1971, 138, 507-511). Dex exerts its anti-inflammatory effects primarily by reducing LPS induced production of pro-inflammatory cytokines such as TNF-α (Waage et al., Immunology 1988, 63, 299-302), IL-6 (Waage et al. European Journal of Immunology 1990, 20, 2439-2443), IL-12p40 (Ma et al., Journal of Immunology 2004, 172, 318-330). Although the mechanism of action is not completely understood, dex regulates transcription as well as the translation of cytokines. By inhibiting NFθB activation and translocation to the nucleus, dex negatively regulates the transcription of proinflammatory cytokines such as IL-6. In human lung fibroblasts, TNF-α induced IL-6 mRNA stability is decreased on dex treatment (Tobler et al., Blood 1992, 79, 45-51). Evidence suggests that dex inhibits TNF-α expression at the translation level (Beutler et al. Science 1986, 232, 977-980; Han et al., Journal of Experimental Medicine 1990, 171, 465-475; Han et al., Journal of Experimental Medicine 1990, 172, 391-394).
Molecules possessing Ankyrin repeats Induced by LPS (MAIL) belongs to the inhibitor protein Iθb family. MAIL was first identified in LPS stimulated cells and shares about 40-44% homology with bcl3 and the IθB proteins (Kitamura et al., FEBS Letters 2000, 485, 53-56). MAIL has since been identified in IL-1β (INAP) induced cells (Haruta et al., Journal of Biological Chemistry 2001, 276, 12485-12488) and MAIL has two splice variants, MAIL-S and MAIL-L (Kitamura et al., FEBS Letters 2000, 485, 53-56). MAIL-S and MAIL-L were also independently discovered and referred to as Iθb-ζ (Yamazaki et al. 2001), and IL-1 inducible nuclear ankyrin repeat protein (INAP) (Haruta et al. 2001) respectively. The human MAIL DNA sequence is provided as SEQ ID NO:2 and the human MAIL-L amino acid sequence is provided as SEQ ID NO:1. Studies involving MAIL over-expression show nuclear localization of the protein (Yamazaki et al. 2001, Haruta et al. 2001). However, in-situ hybridization studies have shown that the intracellular distribution of MAIL is cell type specific. In B lymphocyte rich regions such as the lymphoid follicles and mesenteric lymph nodes in the mouse spleen, MAIL expression is cytoplasmic whereas staining in the macrophages is nuclear (Kitamura et al. Archives of Histology and Cytology 2003, 66, 53-62).
Recent reports analyzing the MAIL promoter suggest that the promoter is induced, in part, by NFκB (Ito et al., Gene 2004, 342, 137-143). MAIL expression is TLR4 dependent. In mice with a mutated tlr4 gene, LPS induced MAIL expression is significantly reduced (Kitamura et al., Journal of Veterinary Medical Science, 2002; Vol. 64, 419-422). Over-expression of MAIL potentiates LPS induced IL-6 expression. MAIL has also been shown to regulate NFθB activity in the nucleus (Kitamura et al. 2000; Haruta et al. 2001; Yamazaki et al. 2001). Cells from MAIL knock-out mice have been shown to have impaired IL-6 production in response to LPS, IL-1βand other TLR agonists. It has been shown that MAIL interacts with the p50 subunit of NFθB and that the MAIL knock-out mice have the same responses to IL-1R/TLR agonists as the NFkB/p50 deficient mice. Abrogation of MAIL expression also resulted in decreased expression of genes such as IL12b and Csf2. MAIL knock-out resulted in decreased expression of LPS induced GM-CSF, IL-12p40, G-CSF, C/EBP-δ and endothelin (Yamamoto et al., Nature 2004, 430, 218-222). MAIL knock-out mice develop severe atopic dermatitis like disease suggesting that MAIL is important for regulation of the immune response (Shiina et al. Immunogenetics 2001, 53, 649-655). Although the exact role of MAIL remains unclear, the protein continues to emerge as an important factor in the regulation of inflammatory responses.
United States patent publication US2002182586, discloses the human MAIL sequence as one of the genes, which may be associated with carcinoma. United States patent publication US2002182586 discloses the use of oncogenic retroviruses, whose sequences insert into the genome of the host organism resulting in carcinoma, allows the identification of host sequences involved in carcinoma. Publication WO0170979 discloses genes associated with ovarian cancer as well as methods of assessing whether a patient is afflicted with ovarian cancer.
There exists a need to identify modulators (agonists, inverse agonists and antagonists) of MAIL. Additionally, there is a need for systems that can be used to test compounds that potentially modulate MAIL gene expression and MAIL protein activity. Identification and testing of such compounds would enable the treatment of various diseases where pro-inflammatory cytokines are implicated, such as inflammation, autoimmune diseases such as Rheumatoid arthritis, Alzheimer's disease, myocardial infarction, Paget's disease, osteoporosis, solid tumors such as renal cell carcinoma, breast, prostate and bladder cancers, certain neurological cancers, as well as B cell malignancies (Trikha et al., Clinical Cancer Research 9, 4653-4665). MAIL antagonists can also be useful in the treatment of chronic inflammatory disorders such as systemic Lupus Erythematosus, Multiple sclerosis, Crohn's disease, transplant rejection, psoriasis, allergic contact dermatitis and other atopic eczemas, rheumatoid arthritis, and chronic inflammatory bowel disease (Asadullah et al., Pharmacological reviews 2003, 55, 241-269).